UV diode laser excitation in flow cytometry

Abstract

The present invention generally relates to a method and apparatus for exciting particles, and more specifically relates to analyzers or sorters for exciting fluorescently labeled particles with a multimode diode laser and the optics for making high resolution determinations from the multimode diode laser beam excitation.

We claim: 
     
       1. An instrument comprising:
 a flow source for delivering sperm along a flow axis to an interrogation zone; 
 a laser that produces a multimode beam along a beam axis which illuminates sperm at the interrogation zone, wherein the multimode beam has a transverse beam profile with multiple spatially separated intensity peaks; 
 beam shaping optics located on the beam axis which compress a beam height and a beam width of the transverse beam profile differently and focus the transverse beam profile to form a manipulated transverse beam profile with a pseudo-Gaussian intensity distribution at the interrogation zone, wherein the beam shaping optics highly compress the beam width, and wherein the manipulated transverse beam profile provides enough uniform laser exposure to sperm at the interrogation zone for sex sorting sperm; 
 a detector for detecting reflected and/or fluoresced light from the illuminated sperm at the interrogation zone and for producing a signal representative of particle characteristics; and 
 a processor in communication with the detector for analyzing the signal and determining particle characteristics. 
 
     
     
       2. The instrument of  claim 1  wherein the beam shaping optics further comprise:
 a first element for altering the beam width of the transverse beam profile; and 
 a second element for altering the beam height of the transverse beam profile. 
 
     
     
       3. The instrument of  claim 2  wherein the first element is located on the beam axis and comprises a cylindrical lens. 
     
     
       4. The instrument of  claim 2  wherein the second element is located on the beam axis and comprises a cylindrical lens. 
     
     
       5. The instrument of  claim 1  wherein the laser produces a multimode beam at a wavelength between 350 nm and 400 nm. 
     
     
       6. The instrument of  claim 5  wherein the laser produces a multimode beam at a wavelength between 370 nm and 380 nm. 
     
     
       7. The instrument of  claim 6  wherein the beam shaping optics comprises a pair of crossed cylindrical lenses. 
     
     
       8. The instrument of  claim 7  wherein each crossed cylindrical lens is oriented 90 relative to the other lens. 
     
     
       9. The instrument of  claim 8  wherein the first cylindrical lens has a focal length between 10 mm and 150 mm. 
     
     
       10. The instrument of  claim 8  wherein the second cylindrical lens has a focal length of between 10 mm and 150 mm. 
     
     
       11. The instrument of  claim 6  wherein the transverse beam profile is focused into a beam spot 26 μm by 160 μm. 
     
     
       12. The instrument of  claim 6  wherein the transverse beam profile is focused into a beam spot 29 μm by 100 μm. 
     
     
       13. The instrument of  claim 6  wherein the laser is oriented 90 degrees about the beam axis. 
     
     
       14. The instrument of  claim 6  further comprising an orientating device for orienting sperm within the flow path. 
     
     
       15. The instrument of  claim 6  further comprising a sorting device for sorting the sperm based upon the determined particle characteristics. 
     
     
       16. The instrument of  claim 15  wherein the sorting devices comprises one selected from the group consisting of: deflection plates, a fluid switching element, and an ablation laser. 
     
     
       17. The instrument of  claim 6  wherein the detector comprises a pair of orthogonal photo detectors. 
     
     
       18. The instrument of  claim 6  wherein the laser comprises a UV multimode diode laser operated at 200 mW. 
     
     
       19. The instrument of  claim 6  wherein the beam shaping optics comprises micro-electro-mechanical elements to redirect all, or portions of, the transverse beam profile to shape the transverse beam profile. 
     
     
       20. The instrument of  claim 6  further comprising a second laser for producing a second multimode beam. 
     
     
       21. The instrument of  claim 20  further comprises beam combining optics for producing a combined beam along the beam axis from the first and the second lasers, wherein the combined beam comprises higher power and more uniform intensity characteristics across the combined transverse beam profile as compared to either the first laser or the second laser alone. 
     
     
       22. A method of processing particles comprising:
 flowing sperm along a fluid flow path having an interrogation zone; 
 producing a multimode beam directed at the interrogation zone, wherein the multimode beam has a transverse beam profile including multiple spatially separated intensity peaks; 
 compressing a beam height and a beam width of the transverse beam profile differently and focusing the transverse beam profile to create a manipulated transverse beam profile with pseudo-Gaussian intensity distribution at the interrogation zone, wherein the beam shaping optics highly compress the beam width, and wherein the manipulated transverse beam profile provides enough uniform laser exposure to sperm at the interrogation zone for sex sorting sperm; 
 illuminating sperm at the interrogation zone with the manipulated beam; 
 detecting reflected and/or fluoresced light from sperm as they pass the interrogation zone and producing a signal; and 
 classifying sperm based upon the produced signals. 
 
     
     
       23. The method of processing sperm according to  claim 22  further comprising:
 selecting sperm based on their classification; and 
 collecting selected sperm. 
 
     
     
       24. The method of processing sperm according to  claim 22  wherein the step of compressing the transverse beam profile further comprises:
 focusing the multimode beam; and 
 shaping the transverse beam profile. 
 
     
     
       25. The method of processing sperm according to  claim 22  wherein the step of compressing the transverse beam profile further comprises:
 compressing the beam height of the transverse beam profile with a first element to a beam height between 10 and 30 micrometers. 
 
     
     
       26. The method of processing sperm according to  claim 25  wherein the step of compressing the transverse beam profile further comprises:
 compressing the beam width of the beam profile to a beam width between 100 micrometers and 200 micrometers. 
 
     
     
       27. The method of processing sperm according to  claim 22  further comprising: modifying the height to width ratio of the beam profile to between 1 to 4 and 1 to 5. 
     
     
       28. The method of processing sperm according to  claim 22  wherein the step of compressing the transverse beam profile further comprises: focusing the multimode beam and varying the aspect ratio of the beam height to the beam width until the multiple spatially separated intensity peaks in the transverse beam profile are reduced or eliminated.